Tissue preparation protocol. Animal were killed by rapid cervical dislocation. Eyes were removed immediately and placed in RNAlater at room temperature. Usually six eyes from animals with a common sex, age, and strain were stored in a single tube.

Each array was hybridized with a pool of RNA from 4 to 8 eyes from 2 to 4 animals. RNA was extracted at UTHSC by Zhiping Jia. If tissue was saved for RNA extraction at a later time, eyes were placed directly in RNAlater (Ambion, Inc.) and treated per the manufacturer’s directions. If eyes were used for immediate RNA extraction then we proceeded immediately to the next steps.

Dissecting and preparing eyes for RNA extraction

1. Place eyes for RNA extraction in RNA STAT-60 (Tel-Test Inc.) and process per manufacturer’s instructions (in brief form below).
2. Store RNA in 75% ethanol at –80 deg. C until use.

Total RNA was extracted with RNA STAT-60 (Tel-Test Inc.) according to the manufacturer's instructions. Briefly we:

1. homogenize tissue samples in the RNA STAT-60 (1 ml/50 to 100 mg tissue)
2. allowed the homogenate to stand for 5 min at room temperature
3. added 0.2 ml of chloroform per 1 ml RNA STAT-60
4. shook the sample vigorously for 15 sec and let the sample sit at room temperature for 3 min
5. centrifuged at 12,000 G for 15 min
6. transfered the aqueous phase to a fresh tube
7. added 0.5 ml of isopropanol per 1 ml RNA STAT-60
8. vortexed and allowed sample to stand at room temperature for 5-10 min
9. centrifuged at 12,000 G for 10-15 min
10. removed the supernatant and washed the RNA pellet with 75% ethanol
11. stored the pellet in 75% ethanol at -80 deg C until use

Sample Processing. Samples were processed in the VA Medical Center, Memphis, Rheumatology Disease Research Core Center lead by Drs. John Stuart and Weikuan Gu. All processing steps were performed by Dr. Yan Jiao. In brief, samples were purified using a standard sodium acetate in alcohol method (recommended by Affymetrix). The RNA quality was checked using a 1% agarose gel. The 18S and 28S bands had to be clear and the 28S band had to be more prominent. RNA concentation was measured using a spectrophotometer. The 260/280 ratios had to be greater than 1.7, and the majority were 1.8 or higher. We used a total of 8 micrograms of RNA as starting amount for cDNA synthesis using a standard Eberwine T7 polymerase method (Superscript II RT, Invitrogen Inc., Affy Part No 900431, GeneChip Expression 3' Amplification One-Cyle cDNA Synthesis Kit). The Affymetrix IVT labeling kit (Affy 900449) was used to generate labeled cRNA. At this point the cRNA was evaluated again using both the 260/280 ratio (values of 2.0 or above were acceptable) and 1% agarose gel inspection of the product (a size range from 200 to 7000 bp is considered suitable for use). We used 45 micrograms of labeled cRNA for fragmentation. Those samples that passed both QC steps (<10% usually fail) were then sheared using a fragmentation buffer included in the Affymetrix GeneChip Sample Cleanup Module (Part No.900371). After fragmentation, samples were either stored at -80 deg. centrigrade until use (one third roughly) or were immediately used for hybridization.

Dealing with Ocular Pigmentation: Variable ocular pigmentation is a potential confound in a study of the whole eye transcriptome. Even the most careful RNA preparations of brown and beige colored mice tend to have faint residual pigmentation that does affect hybridization signal. The key determinant of this interesting effect is the Tyrp1 (brown) locus on Chr 5 at about 80 Mb. Loci on Chr 4 that map at this location should be considered with skepticism and reviewed carefully. To address this problem Yan Jiao purified total RNA a second time using the Qiagen RNeasy MinElute Cleanup Kit (Cat No. 74204). This was done for 8 colored samples (R2534E2, R2578E2, R1441E2, R2537E2, R2536E2, R2589E2, R2539E2), of which 5 were finally included in this data set (cases in which the strain ID is labeled with asterisks in the table).

Replication and Sample Balance: Our goal is to obtain data for independent biological sample pools from at least one of sample from each sex for all BXD strains. The current first batch of array data, represents a balanced sample of males and females, but without within-strain replication. We expect to add roughly 100 additional samples inthe next few months.

Batch Structure: This data set consists of a single batch. The great majority of arrays are from a single lot.

The table below summarizes information on strain, age, sex, original CEL filename, several quality control values, and source of mice. Columns labeled "PDNN 2Z outlier" and "RMA 2Z outlier" list the fraction of probe sets with values that deviated more than 2 z units from the mean. Scale factor, background average, present, absent, marginal and 3'/5' ratios for actin and Gapdh were collated from the Affymetrix Report (RPT) files.

Id tube ID group type Strain age sex original CEL filename PDNN 2Z outlier RMA 2Z outlier scale factor background average present absent marginal AFFX-b-ActinMur(3'/5') AFFX-GapdhMur(3'/5') source 1 R2607E1 GDP BXD C57BL/6J 67 F R2605E.CEL 0.005 0.009 2.428 115.12 0.586 0.4 0.014 1.31 0.76 UTM RW 2 R0872E2 GDP BXD C57BL/6J 66 M R0872E.CEL 0.013 0.012 3.128 88.58 0.589 0.396 0.015 1.3 0.79 UTM RW 3 R2572E1 GDP BXD DBA/2J 65 M R2572E.CEL 0.041 0.051 2.406 79.07 0.555 0.429 0.016 1.37 0.79 UTM RW 4 R2601E1 GDP BXD B6D2F1 73 F R2601E.CEL 0.003 0.004 2.545 91.96 0.589 0.396 0.015 1.44 0.78 UTM RW 5 R2602E1 GDP BXD B6D2F1 73 M R2602E.CEL 0.001 0.004 2.599 84.44 0.597 0.388 0.015 1.37 0.78 UTM RW 6 R2600E1 GDP BXD D2B6F1 72 F R2600E.CEL 0.003 0.008 2.47 94.75 0.581 0.402 0.017 1.41 0.78 UTM RW 7 R2604E1 GDP BXD D2B6F1 69 M R2604E.CEL 0.003 0.007 2.657 89.63 0.594 0.392 0.015 1.28 0.79 UTM RW 8 R2597E1 BXD BXD2 61 M R2597E.CEL 0.003 0.007 2.374 93.56 0.603 0.383 0.015 1.34 0.77 Glenn 9 R2591E1 BXD BXD5 60 F R2591E.CEL 0.051 0.009 1.7 136.48 0.585 0.4 0.015 1.33 0.78 Glenn 10 R2570E1 BXD BXD6 65 F R2570E.CEL 0.002 0.006 1.987 86.73 0.585 0.4 0.015 1.46 0.76 UTM RW 11 R2538E1 BXD BXD8 77 F R2538E.CEL 0.037 0.028 1.905 101.98 0.612 0.373 0.015 1.52 0.79 UTM RW 12 R2569E1 BXD BXD9 67 M R2569E.CEL 0.014 0.027 1.753 87.36 0.551 0.434 0.015 2.82 3.14 UTM RW 13 R2581E1 BXD BXD11 65 F R2581E.CEL 0.006 0.012 1.941 88.55 0.621 0.364 0.016 1.55 0.81 UTM RW 14 R2543E1 BXD BXD12 63 M R2543E.CEL 0.036 0.007 1.605 117.69 0.586 0.399 0.016 1.43 0.77 UTM RW 15 R2586E1 BXD BXD13 60 F R2586E.CEL 0.020 0.035 2.006 73.61 0.564 0.42 0.016 2.85 3.81 Glenn 16 R2557E1 BXD BXD14 60 F R2557E.CEL 0.014 0.017 1.83 98.76 0.625 0.361 0.014 1.31 0.78 Glenn 17 R2567E1 BXD BXD16 60 M R2567E.CEL 0.016 0.025 2.239 82.35 0.567 0.416 0.017 1.37 0.75 Glenn 18 R2559E1 BXD BXD18 59 M R2559E.CEL 0.035 0.006 1.654 103.68 0.608 0.377 0.015 1.27 0.78 Glenn 19 R2560E1 BXD BXD19 60 F R2560E.CEL 0.026 0.007 1.792 98.33 0.609 0.375 0.016 1.35 0.8 Glenn 20 R2584E1 BXD BXD20 59 F R2584E.CEL 0.003 0.007 2.07 83.82 0.593 0.391 0.016 1.4 0.76 Glenn 21 R2541E2 BXD BXD21 61 M R2541E2.CEL 0.049 0.036 2.625 125.08 0.56 0.424 0.015 1.29 0.78 UTM RW 22 R2553E1 BXD BXD22 58 F R2553E.CEL 0.003 0.005 1.952 111.3 0.599 0.385 0.015 1.28 0.76 Glenn 23 R2558E1 BXD BXD23 60 F R2558E2.CEL 0.013 0.015 1.908 114.53 0.599 0.388 0.014 1.2 0.82 Glenn 24 R2589E2 BXD BXD24-rd* 59 M R2589E2.CEL 0.098 0.098 2.606 112.19 0.575 0.409 0.016 1.24 0.8 Glenn 25 R2573E1 BXD BXD25 67 F R2573E2.CEL 0.009 0.018 3.153 71.88 0.579 0.407 0.014 1.77 0.97 UAB 26 R2562E1 BXD BXD28 60 F R2562E.CEL 0.003 0.005 1.649 116.35 0.599 0.384 0.017 1.37 0.79 Glenn 27 R2561E1 BXD BXD29 60 F R2561E.CEL 0.019 0.029 1.952 93.32 0.583 0.402 0.015 2.19 1 Glenn 28 R2598E1 BXD BXD31 61 M R2598E.CEL 0.003 0.006 1.989 106.48 0.609 0.376 0.015 1.27 0.78 UTM RW 29 R2563E1 BXD BXD32 63 F R2563E.CEL 0.008 0.011 1.547 101.52 0.619 0.367 0.014 1.5 0.8 UTM RW 30 R2542E1 BXD BXD33 67 F R2542E.CEL 0.010 0.016 2.128 97.08 0.565 0.418 0.016 1.91 0.93 UTM RW 31 R2585E1 BXD BXD34 60 M R2585E.CEL 0.007 0.014 2.64 75.13 0.583 0.4 0.017 1.25 0.77 Glenn 32 R2532E1 BXD BXD38 62 M R2532E.CEL 0.002 0.003 2.038 93.65 0.598 0.387 0.015 1.37 0.8 UTM RW 33 R2574E1 BXD BXD39 70 F R2574E.CEL 0.001 0.004 1.981 90.64 0.612 0.373 0.015 1.39 0.78 UTM RW 34 R2590E1 BXD BXD40 60 M R2590E.CEL 0.004 0.007 2.708 77.3 0.591 0.393 0.015 1.4 0.77 Glenn 35 R2596E1 BXD BXD42 59 M R2596E.CEL 0.013 0.017 2.632 108.46 0.59 0.396 0.015 1.24 0.8 Glenn 36 R2605E1 BXD BXD43 79 M R2607E.CEL 0.003 0.006 1.817 131.22 0.605 0.382 0.013 1.32 0.8 UTM RW 37 R2594E1 BXD BXD44 63 F R2594E.CEL 0.004 0.009 1.766 117.33 0.598 0.388 0.014 1.35 0.85 UTM RW 38 R2592E1 BXD BXD45 62 M R2592E.CEL 0.002 0.004 1.85 106.16 0.601 0.386 0.013 1.43 0.85 UTM RW 39 R2606E1 BXD BXD48 78 M R2606E.CEL 0.003 0.010 2.556 106.16 0.589 0.397 0.014 1.35 0.83 UTM RW 40 R2603E1 BXD BXD51 66 F R2603E.CEL 0.003 0.009 2.488 115.16 0.577 0.408 0.015 1.24 0.79 UTM RW 41 R2534E2 BXD BXD61* 70 F R2534E2.CEL 0.030 0.028 2.473 117.76 0.579 0.406 0.015 1.42 0.79 UTM RW 42 R2611E1 BXD BXD64 68 M R2611E.CEL 0.013 0.022 2.292 91.99 0.58 0.405 0.015 1.57 1.06 UTM RW 43 R2583E1 BXD BXD65 60 M R2583E.CEL 0.005 0.010 2.492 70.43 0.569 0.415 0.016 1.67 1.01 UTM RW 44 R2536E2 BXD BXD66* 64 F R2536E2.CEL 0.039 0.065 2.74 108.62 0.561 0.423 0.017 1.28 0.79 UTM RW 45 R2551E1 BXD BXD68 67 F R2551E.CEL 0.037 0.039 2.493 92.38 0.543 0.441 0.016 2.91 1.55 UTM RW 46 R2593E1 BXD BXD69 59 F R2593E.CEL 0.008 0.013 1.672 127.6 0.592 0.395 0.013 1.47 0.92 UTM RW 47 R2537E2 BXD BXD70* 59 M R2537E2.CEL 0.046 0.044 2.93 98.66 0.58 0.405 0.016 1.29 0.75 UTM RW 48 R2565E1 BXD BXD75 61 F R2565E.CEL 0.009 0.017 1.79 101.68 0.58 0.405 0.015 2.31 3.47 UTM RW 49 R2579E1 BXD BXD80 65 F R2579E.CEL 0.005 0.010 2.419 72.13 0.592 0.394 0.015 1.73 0.82 UTM RW 50 R2540E1 BXD BXD87 63 M R2540E.CEL 0.013 0.016 2.333 93.15 0.611 0.374 0.014 1.22 0.81 UTM RW 51 R2545E1 BXD BXD89 67 M R2546E.CEL 0.046 0.046 1.667 104.76 0.562 0.423 0.015 3.6 9.84 UTM RW 52 R2578E2 BXD BXD90* 61 F R2578E2.CEL 0.033 0.034 2.785 92.27 0.586 0.398 0.016 1.52 0.77 UTM RW 53 R2554E1 BXD BXD96 67 M R2554E.CEL 0.004 0.004 2.177 93.02 0.602 0.383 0.015 1.46 0.77 UTM RW 54 R2577E1 BXD BXD97 55 M R2577E.CEL 0.019 0.016 2.07 76.58 0.595 0.391 0.014 1.87 1.29 UTM RW 55 R2595E1 GDP 129S1/SvImJ 59 F R2595E.CEL 0.017 0.021 1.792 115.39 0.61 0.375 0.015 1.46 0.77 UTM RW 56 R2533E1 GDP 129S1/SvImJ 60 M R2533E.CEL 0.021 0.013 2.107 93.55 0.579 0.405 0.016 1.37 0.78 UTM RW 57 R2546E1 GDP A/J 66 F R2545E.CEL 0.018 0.014 1.989 95.59 0.586 0.397 0.017 1.47 0.78 UTM RW 58 R0754E2 GDP A/J 60 M R0754E.CEL 0.014 0.016 2.718 85.63 0.598 0.387 0.015 1.36 0.76 JAX 59 R1676E1 GDP BALB/cByJ 83 F R1676E.CEL 0.042 0.041 2.685 98.37 0.589 0.396 0.015 1.46 0.74 JAX 60 R1672E1 GDP BALB/cByJ 83 M R1672E.CEL 0.022 0.022 2.216 110.52 0.599 0.386 0.015 1.26 0.8 JAX 61 R1700E1 GDP C3H/HeJ 83 F R1700E.CEL 0.090 0.092 2.978 68.77 0.608 0.379 0.014 1.48 0.78 UTM RW 62 R1704E1 GDP C3H/HeJ 83 M R1704E.CEL 0.086 0.089 2.581 88.29 0.601 0.386 0.013 1.38 0.84 UTM RW 63 R2564E1 GDP CAST/Ei 64 F R2564E.CEL 0.078 0.064 1.937 88.89 0.585 0.399 0.016 1.6 0.77 JAX 64 R2580E1 GDP CAST/Ei 64 M R2580E.CEL 0.076 0.067 2.089 94.64 0.582 0.401 0.017 1.4 0.76 JAX 65 R2636E1 GDP KK/HIJ 64 F R2636E.CEL 0.023 0.026 2.61 93.1 0.589 0.395 0.015 1.39 0.76 UTM RW 66 R2637E1 GDP KK/HIJ 64 M R2637E.CEL 0.039 0.020 2.189 102.78 0.594 0.39 0.015 1.3 0.79 UTM RW 67 R0999E1 GDP LG/J 57 F R0999E.CEL 0.012 0.012 2.448 82.09 0.594 0.391 0.015 1.38 0.79 UTM RW 68 R1004E1 GDP LG/J 65 M R1004E.CEL 0.013 0.015 2.438 91.71 0.587 0.398 0.015 1.38 0.79 UTM RW 69 R1688E1 GDP NOD/LtJ 66 F R1688E.CEL 0.017 0.019 2.664 97.65 0.586 0.399 0.015 1.26 0.8 JAX 70 R2566E1 GDP NOD/LtJ 76 M R2566E2.CEL 0.019 0.025 3.031 69.44 0.598 0.388 0.015 1.38 0.75 UTM RW 71 R2550E1 GDP NZO/HlLtJ 96 M R2550E.CEL 0.023 0.015 1.794 87.16 0.607 0.378 0.015 1.52 0.82 JAX 72 R2535E1 GDP NZO/HlLtJ 62 F R2535E.CEL 0.046 0.025 1.893 85.67 0.604 0.382 0.014 1.41 0.85 JAX 73 R2634E1 GDP PWD/PhJ 62 F R2635E.CEL 0.077 0.069 3.292 89.8 0.559 0.425 0.016 1.57 0.81 JAX 74 R2635E1 GDP PWD/PhJ 62 M R2634E.CEL 0.088 0.081 3.722 80.05 0.542 0.441 0.017 1.53 0.85 JAX 75 R2544E1 GDP PWK/PhJ 63 F R2544E.CEL 0.106 0.100 2.196 107.51 0.549 0.435 0.017 1.36 0.82 JAX 76 R2549E1 GDP PWK/PhJ 83 M R2549E.CEL 0.065 0.048 2.275 83.8 0.573 0.412 0.015 1.57 0.83 JAX 77 R2368E1 GDP WSB/EiJ 67 F R2368E.CEL 0.025 0.028 2.567 85.7 0.595 0.391 0.014 1.29 0.74 UTM RW 78 R2547E1 GDP WSB/EiJ 67 M R2547E.CEL 0.032 0.021 2.135 90.04 0.582 0.401 0.016 1.32 0.77 UTM RW